ARTICLE https://doi.org/10.1038/s41467-021-24866-3 OPEN PALI1 facilitates DNA and nucleosome binding by PRC2 and triggers an allosteric activation of catalysis Qi Zhang1,3, Samuel C. Agius1,3, Sarena F. Flanigan1, Michael Uckelmann1, Vitalina Levina1, Brady M. Owen1 & ✉ Chen Davidovich 1,2 1234567890():,; The polycomb repressive complex 2 (PRC2) is a histone methyltransferase that maintains cell identities. JARID2 is the only accessory subunit of PRC2 that known to trigger an allosteric activation of methyltransferase. Yet, this mechanism cannot be generalised to all PRC2 variants as, in vertebrates, JARID2 is mutually exclusive with most of the accessory subunits of PRC2. Here we provide functional and structural evidence that the vertebrate- specific PRC2 accessory subunit PALI1 emerged through a convergent evolution to mimic JARID2 at the molecular level. Mechanistically, PRC2 methylates PALI1 K1241, which then binds to the PRC2-regulatory subunit EED to allosterically activate PRC2. PALI1 K1241 is methylated in mouse and human cell lines and is essential for PALI1-induced allosteric activation of PRC2. High-resolution crystal structures revealed that PALI1 mimics the reg- ulatory interactions formed between JARID2 and EED. Independently, PALI1 also facilitates DNA and nucleosome binding by PRC2. In acute myelogenous leukemia cells, overexpression of PALI1 leads to cell differentiation, with the phenotype altered by a separation-of-function PALI1 mutation, defective in allosteric activation and active in DNA binding. Collectively, we show that PALI1 facilitates catalysis and substrate binding by PRC2 and provide evidence that subunit-induced allosteric activation is a general property of holo-PRC2 complexes. 1 Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, VIC, Australia. 2 EMBL-Australia and the ARC Centre of Excellence in Advanced Molecular Imaging, Clayton, VIC, Australia. 3These authors ✉ contributed equally; Qi Zhang, Samuel C. Agius. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:4592 | https://doi.org/10.1038/s41467-021-24866-3 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24866-3 ver the course of evolution, gene families tend to a paralogue of PALI1, termed PALI2, encoded by the LCORL Oexpand1. Accordingly, the number of genes linked to the locus. PALI1 is required for mouse development18 and promotes same function is commonly increased in vertebrates with the histone methyltransferase (HMTase) activity of PRC2 in vitro respect to invertebrates, especially in cases of genes coding for and in vivo16,18, but the molecular mechanism is unknown. transcriptional regulators2,3. For instance, the histone H3K4 Here, we show that PALI1 allosterically activates PRC2 and methyltransferase MLL/COMPASS complex expended from one facilitates substrate binding. Mechanistically, PALI1 lysine 1241 is in yeast to three sub-types in fly and six complexes in human, a substrate for PRC2 in vitro and is methylated in multiple where additional subunits emerged through each expansion4. human and mouse cell lines. Once in a di- or tri-methyl-form, Similar expansion took place for the histone ubiquitin ligase PALI1-K1241me2/3 binds to the regulatory subunit EED and polycomb repressive complex 1 (PRC1): from two complexes in allosterically activates PRC2. Structural and functional evidence the fly to at least six variants in vertebrates5,6. Vertebrate-specific indicates that PALI1 has emerged through a convergent evolution subunits of histone modifiers provide the opportunity to identify to mimic the function of JARID2 within the context of the molecular mechanisms that are fundamental to chromatin biol- PRC2.1 complex. We also show that the PRC2-binding domain of ogy and, therefore, re-emerged through the course of evolution. PALI1 increases the affinity of PRC2 to DNA by >25-fold and to The polycomb repressive complex 2 (PRC2) is a histone mononucleosome substrates by >50-fold. Allosteric activation methyltransferase complex that is required for the maintenance of and chromatin binding are two separate functions of PALI1, cell identity in all multicellular organisms. At the molecular level, demonstrated by a separation-of-function PALI1 mutant: defec- PRC2 maintains the repressed state of developmentally expressed tive in allosteric activation but active in substrate binding. genes through the tri-methylation of lysine 27 in histone H3 Overall, our results reveal how PRC2 is regulated by PALI1 at the (H3K27me3), a hallmark of facultative heterochromatin6,7. The molecular level, with a positive regulation at the level of both core PRC2 complex includes four subunits7–10, but it has a low catalysis and substrate binding. More broadly, our data implies histone methyltransferase activity and low affinity to DNA. that subunit-induced allosteric activation of PRC2 is an indis- Therefore, holo-PRC2 complexes include additional protein pensable molecular property that is permitted in most sub-types subunits—termed accessory subunits. Most of the accessory of vertebrate holo-PRC2 complexes. subunits of the vertebrate PRC2 emerged through gene duplica- tion and some are vertebrate specific, with the latter poorly understood mechanistically. RESULTS The accessory subunits are collectively required for the PALI1 K1241 is methylated in mouse and human cell lines.Ina recruitment of PRC2 to chromatin and for the regulation of its search for a PRC2.1 accessory subunit that could trigger an enzymatic activity11,12. Unbiased proteomic studies13–17 revealed allosteric activation of PRC2, we first set out to map the PRC2 two distinct holo-PRC2 complexes—PRC2.1 and PRC2.2— methylome in mouse and human cells. We reasoned that affinity defined by their mutually exclusive accessory subunits16. The purification of PRC2 followed by tandem mass spectrometry (AP- PRC2.2 complex is nearly identical in fly and human, and MS) will allow for the identification of methyl-lysines residing includes the accessory subunits AEBP2 and JARID2. Contrarily to within assembled PRC2 complexes in vivo. Hence, we analysed PRC2.2, the PRC2.1 complex went through a massive expansion multiple publicly available liquid chromatography with tandem over evolution: from one accessory subunit in fly, to at least five in mass spectrometry (LC-MS/MS) data originating from AP-MS vertebrates13–17. The fly PRC2.1 accompanies a single accessory experiments, where PRC2 subunits were used as baits15,26–29 subunit: Pcl. The vertebrate PRC2.1 is far more complex: it (Fig. 1a and Supplementary Data 1). Although these contains one of the three polycomb-like (PCL) proteins16 (PHF1, studies15,26–29 were not focused on the methylation of MTF2 or PHF19) together with either EPOP13,16 or PALI116,18 PRC2 subunits, the high quality of the raw data allowed us to (also annotated as LCOR-CRA_b, LCOR isoform 3 or detect methyl-lysines in tryptic peptides (Supplementary Data 1). C10ORF12). Recent works indicate some non-redundant func- As expected, the PRC2 methylome contains the previously tions of the PRC2.1 and PRC2.2 complexes in mouse embryonic reported methylations in JARID2 K11621 and EZH2 K514 and cells11,12, but the molecular basis is unknown. K51530–32. The PRC2.2-specific subunit JARID2 has two activities that The two most frequently detected di- and tri-methyl lysines in were implicated in nucleating H3K27me3: chromatin binding19,20 the accessory subunits of PRC2 were in JARID2 K116 and PALI1 and allosteric stimulation of histone methyltransferase K1241 (Fig. 1a), with the former triggering an allosteric activation (HMTase)21. During JARID2-induced allosteric activation, PRC2 of PRC221. Specifically, PALI1 K1241 is methylated in five of the first di- or tri-methylates lysine 116 in JARID2 (JARID2- seven cell lines that were tested, including both human and K116me2/3). Next, the di/tri-methyl-lysine binds to the reg- mouse cell lines: HEK293T (human embryonic kidney), STS26T ulatory subunit EED and triggers an allosteric activation of (human malignant peripheral nerve sheath tumour), LnCAP PRC221. This mechanism is thought as a “jump-start” to activate (human prostate cancer), U2OS (human osteosarcoma) and PRC28. After the nucleation of H3K27me3, histone tails carrying mouse embryonic stem cells (mESC). In four of these cell lines, the H3K27me3 mark bind to the regulatory subunit EED22 to PALI1 K1241 was identified either in its di- or tri-methyl form trigger further allosteric activation of PRC223. Yet, knockout of (i.e. PALI1 K1241me2/3). JARID2 in mouse ESC cells lacks major effect on H3K27me3 In some of the cell lines, we also identified methylations in globally and locally11,21. This implies a parallel role taken by PALI1 K1214 and K1219, in agreement with a previous PRC2.1 during de novo introduction of H3K27me3, in agreement proteomic analysis in HCT116 cells30. The same study also with the PRC2.1-specific subunit MTF2 being essential for this identified methylations of EZH2 K510 and K515 that have more process11,24. Yet, a mechanism for subunit-induced allosteric recently been shown to regulate PRC231,32. Yet, K1241 was not activation of the PRC2.1 complex is yet to be discovered. identified in that study30, which used antibodies against methyl- Multiple unbiased proteomic studies identified C10ORF12 as lysines for immunoaffinity purification ahead of the mass an accessory subunit of PRC2.115,16,25 and, thus, mutually spectrometry30.